Pyrrolidone carboxylate amino acids

Pyrrolidone carboxylyl peptidase activity has been followed using pyrrolidone carboxylyl L-alanine (or other pyrrolidone carboxylyl-amino acids) as substrate by following the amount of amino acid released as determined by the ninhydrin method (137). In another assay procedure the release of -naphthyl amine from L-pyrrolidone carboxylic-/ -naphthylamide (135) is determined by the diazotization procedure of Bratton and Marshall (138) as modified by Goldberg and Rutenberg (139). 

This enzyme [EC 3.4.19.3], a member of the C15 peptidase family, is also known as pyroglutamyl-peptidase 1,5-oxoprolyl-peptidase, pyrrolidone-carboxylate peptidase, and pyroglutamyl aminopeptidase. This hydrolase catalyzes the conversion of a 5-oxoprolyl-peptide to produce 5-oxoproline and a peptide. The enzyme will not act on the 5-oxoprolyl peptide if the adjacent amino acid is l-proline. Enzyme activity is inhibited by thiol-blocking reagents. 

It is known that peptides and proteins exhibit a higher absorbance at wavelengths below 240 nm than the component amino acids (40, 41) and that the absorbance at 205 nm of peptides and proteins is approximately proportional to the number of peptide bonds. Pyrrolidone carboxylic acid, which has an internal peptide bond, exhibits this type of absorbance, which may be used under appropriate conditions for its quantitative determination. Thus, Orlowski et al. (13) followed the activity of y-glutamyl cyclotransferase by this procedure after incubation of the enzyme with y-glutamyl amino acids, the reaction mixture was passed through a small column of Dowex 50 (H+) in order to remove interfering substances, and the absorbance of the eluate at 205 nm was then determined. 

Using the data of Wilson and Cannan (18), Cleaves (81) was able to show that the rate of formation of pyrrolidone carboxylic acid from glutamic acid in aqueous solution depends directly on the concentration of the ionic species of glutamic acid in solution. Thus, the reactive species are (I), (II), and (IV), while (III) is relatively unreactive. Protonation of the amino group and dissociation of the y-carboxyl group thus makes these groups less reactive carboxylate ion resonance apparently hinders nucleophilic attack by the amino nitrogen. 

VII. Enzymic Formation of Pyrrolidone Carboxylic Add from y-Glutamyl Amino Acids... 

Fig. 1. The y-glutamyl cycle. I, y-Glutamylcysteine synthetase II, glutathione synthetase III, y-glutamyl transpeptidase IV, y-glutamyl cyclotransferase V. peptidase. PCA = pyrrolidone carboxylic acid AA = amino acid.

The lack of filaggrin and keratohyaline in IV horny layer results in a deficiency of the natural moisturizing factor (NMF) — composed of urocanic acid and pyrrolidone carboxylic acid (PCA) — that is, breakdown products of amino acids in profilaggrin.12 In XRI, on the other hand, there is... 

Scott, I., Harding, C., and Barrett, J., Histidine-rich protein of the keratohyalin granules source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum comeum, Biochim. Biophys. Acta, 719, 110, 1982. 

Tabachnick, J. and Labadie, J.H. Studies on the biochemistry of epidermis. IV. The free amino acids, ammonia, urea and pyrrolidone carboxylic acid content of conventional and germ free albino guinea pig epidermis, J. Invest. Dermatol., 54, 24, 1970. 

Another reaction which occurs readily at acid pH values is transformation of amino-terminal glutamine into amino-terminal pyrrolidone carboxylic acid. 

This was first foimd by Sanger et al. (1955) in a peptide from insulin and was observed with other peptides by Hirs et al. (1956) and Smyth et al. (1962). The reaction appears to occur when acidic buffers or dilute acids are employed for isolation of peptides. Conversion of the cyclic pyrrolidone carboxyl residue to a glutamyl residue is obtained on mild hydrolysis in dilute acids or alkalies. The cyclization reaction leads to difficulties when sequence methods are used which proceed from the amino-terminal end of a peptide. In addition, this reaction can occur when an internal glutamine residue becomes amino-terminal in the course of stepwise sequence analysis under acidic conditions, as in the Edman methods. An incorrect sequence for a peptide from ribonuclease was deduced as the result of cyclization of amino-terminal glutamine and acidic destruction of serine and threonine in the same peptide (Smyth et al., 1962). 

Substances related to MSG and purine 5 -ribonucleotides include peptides, amino acids (e.g. cysteine, homocysteine, cysteine S-sulfonic acid, aspartic acid, a-amino adipic acid, a-methyl glutamic acid, tricholomic acid, ibotenic acid), pyrrolidone carboxylic acid, 3-methyl thiopropyl amine, and others [2, 10], They are of less commercial interest than MSG, IMP, and GMP. Chemical structures of some of these substances are depicted in Fig. 3.53. Relative umami effects of some are shown in Tab. 3.49. Tricholomic acid and ibotenic acid have been found in the mushrooms Tricholoma muscarium and Amanita stroboliformis, respectively. 

A cyclic, internal amide derivative of glutamic acid is pyrrolidone carboxylic acid (also known as pyroglu-tamic acid or 2-oxoproline). Some proteins (e.g., heavy chains of immunoglobulins Chapter 35) and peptides (e.g., thyrotropin-releasing hormone Chapter 33) have py-roglutamic acid as their N-terminal amino acid residue. 

Pyrrolidone carboxylic acid is the N-terminal amino acid residue of the /3-chain of fibrinogen from several species (229,230,242). The amino acid sequence containing pyrrolidone carboxylic acid (Glu) is (243) ... 

A serious limitation of this powerful analytical technique is the inability to detect and quantify Gin and Asn m an amino acid mixture In the acid-catalyzed esterification reaction, the amides (Gin and Asn) are rapidly deaminated and converted to the same derivatives as those formed by Glu and Asp, respectively. Consequently, summations of both Glu and Gin concentrations and Asp and Asn concentrations are determined by this method. It has been shown that when Gin is heated to a temperature of about 100°C in an acidified alcoholic medium, rapid conversion to glutamic acid diester takes place. Pyrrolidone carboxylic acid ester has been shown by mass spectrometry to be a cyclized intermediate (Fig 2). This intermediate reaches its maximum concentrahon after heating at 100°C for 5-10 mm (Collins and Summer, 1978). 

Finally, it should be noted that the distribution of Vh subgroups in mn may be influenced to some extent by the ease with which amino acid sequencing can be accomplished. Since the VhI and VhII subgroups are blocked (pyrrolidone-5-carboxylic acid at the NHg-terminus), there are likely to be more sequences available for VhIII heavy chains. Thus we may have to wait for more extensive serology to be done before the exact distribution of subgroups or subsubgroups in man is well established. 

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